JP7049761B2 - Cell-derived viral vaccine containing low levels of residual cellular DNA treated with β-propiolactone - Google Patents

Abstract

The present invention relates to vaccine products for the treatment or prevention of viral infections. Further provided are methods of reducing contaminants associated with the preparation of cell culture vaccines. Residual functional cell culture DNA is degraded by treatment with a DNA alkylating agent, such as ²-propiolactone (BPL), thereby providing a vaccine comprising immunogenic proteins derived from a virus propagated on cell culture, substantially free of residual functional cell culture DNA.

Description

All literature and online information cited herein are incorporated by reference in their entirety.

Technical Fields The present invention provides improved cell culture products and processes with reduced impurities. Specifically, the invention provides an improved method of degrading any residual functional cell culture DNA associated with a product made in cell culture. According to the present invention, residual functional cell culture DNA is degraded by treatment with a DNA alkylating agent, such as β-propiolactone (BPL). This process can be used to process a series of cell culture products containing vaccines and recombinant proteins.

Commercial production of viral vaccines typically requires large amounts of virus as an antigen source. Commercial quantities of virus for vaccine production can be achieved by culturing and replicating the seed virus in a cell culture system. Suitable cell culture systems for viral replication include mammalian, avian or insect cells, where mammalian cell culture systems ensure proper glycosylation and folding of viral antigenic proteins. Is particularly preferable. For similar reasons, mammalian cell culture systems are also preferred for recombinant protein expression.

When unmodified from their native state, cell cultures have limited replication capacity and are subsequently impractical for producing the required amount of material for commercial vaccines or recombinant proteins. And it becomes insufficient. Therefore, for manufacturing purposes, it is preferred to modify the cells to be "persistent" or "immortalized" cell lines in order to increase the number of times they can divide. Many of these modifications use mechanisms similar to those associated with carcinogenic cells. Therefore, there is a concern that any residual material from the cell culture process (eg, host cell DNA) will be removed from the final product of the vaccine or recombinant protein product produced in these systems.

The standard method for removing residual host cell DNA is by DNase treatment. A convenient method of this type is disclosed in Patent Documents 1 and 2, a two-step process using first a DNase (eg, Benzonase) and then a cationic detergent (eg, CTAB). including.
Current efforts to reduce this risk have focused on reducing the total concentration of residual host cell DNA. An object of the present invention is to further reduce the risk by eliminating the functionality of any residual host cell DNA.

European Patent No. 0870508 U.S. Pat. No. 5,948,410

INDUSTRIAL APPLICABILITY The present invention provides improved cell culture products and processes with reduced impurities. Specifically, the invention provides an improved method of degrading any residual functional cell culture DNA associated with a product made in cell culture. According to the present invention, residual functional cell culture DNA is degraded by treatment with a DNA alkylating agent, such as β-propiolactone (BPL). This process can be used to process a series of cell culture products containing vaccines and recombinant proteins.

The present invention comprises a vaccine comprising an immunogenic protein derived from a virus propagated in a cell culture, wherein the vaccine is substantially free of residual functional cell culture DNA. Furthermore, the present invention relates to a recombinant protein expressed in a cell culture, wherein the final recombinant protein preparation is substantially free of residual functional cell culture DNA.

The functionality of any residual host cell DNA can be eliminated by processing the DNA with an alkylating agent that cleaves the DNA into sufficiently small portions, thus encoding functional proteins, in the chromosomes of human recipients. It cannot be introduced into or otherwise recognized by the recipient DNA replication mechanism. Preferably, the length of the degraded residual cell culture DNA is less than 500 base pairs. More preferably, the length of the degraded residual cell culture DNA is less than 200 base pairs.
The present invention provides, for example, the following items:
(Item 1)
A vaccine comprising an immunogenic protein derived from a virus propagated in a cell culture, wherein the vaccine is substantially free of residual functional cell culture DNA.
(Item 2)
The vaccine according to item 1, wherein any residual cell culture DNA is degraded by treatment with an alkylating agent.
(Item 3)
The vaccine according to item 2, wherein the alkylating agent is β-propiolactone (BPL).
(Item 4)
The vaccine according to item 1, wherein the length of the degraded residual cell culture DNA is less than 500 base pairs.
(Item 5)
The vaccine according to item 1, wherein the length of the degraded residual cell culture DNA is less than 200 base pairs.
(Item 6)
The vaccine according to item 3, wherein the residual cell culture DNA is degraded by treatment with less than 0.1% β-propiolactone (BPL).
(Item 7)
The vaccine according to item 1, wherein the vaccine exhibits a reduced level of aggregation.
(Item 8)
A vaccine containing an immunogenic protein derived from a virus propagated in a cell culture, wherein the residual cell culture DNA is less than 500 base pairs in length.
(Item 9)
The vaccine according to item 8, wherein the residual cell culture DNA is degraded by treatment with an alkylating agent.
(Item 10)
9. The vaccine according to item 9, wherein the alkylating agent is β-propiolactone (BPL).
(Item 11)
Item 8. The vaccine according to item 8, wherein the residual cell culture DNA is less than 300 base pairs in length.
(Item 12)
Item 8. The vaccine according to item 8, wherein the residual cell culture DNA is less than 200 base pairs in length.
(Item 13)
The vaccine according to item 8, wherein the residual cell culture DNA is degraded by treatment with less than 0.1% β-propiolactone (BPL).
(Item 14)
The vaccine according to item 8, wherein the vaccine exhibits a reduced level of aggregation.
(Item 15)
The vaccine according to item 10, which contains less than 0.01% of the combined free propionic acid and β-propiolactone (BPL).
(Item 16)
A method for preparing a vaccine containing an immunogenic protein derived from a virus propagated in a cell culture, the following:
A method comprising (i) degrading residual functional cell culture DNA; and (ii) isolating the immunogenic protein.
(Item 17)
The method of item 16, wherein the step of degrading the residual functional cell culture DNA comprises treatment of the functional cell culture DNA with an alkylating agent.
(Item 18)
17. The method of item 17, wherein the alkylating agent is β-propiolactone (BPL).
(Item 19)
18. The method of item 18, wherein the cell culture DNA is degraded by treatment with less than 0.1% β-propiolactone (BPL).
(Item 20)
The method of item 16, wherein the vaccine exhibits reduced levels of aggregation.
(Item 21)
The method of any one of items 16-20, wherein step (ii) comprises separating DNA from the virion.
(Item 22)
The vaccine according to item 1 or 8, wherein the virus is an influenza virus.
(Item 23)
The cell cultures are MDCK cells, Vero cells, and PER. C. The vaccine according to item 1 or 8, which is selected from the group consisting of 6 cells.
(Item 24)
A product containing a recombinant protein expressed in a cell culture, wherein the product substantially does not contain residual functional cell culture DNA.
(Item 25)
24. The formulation according to item 24, wherein any residual cell culture DNA has been degraded by treatment with an alkylating agent.
(Item 26)
25. The preparation according to item 25, wherein the alkylating agent is β-propiolactone (BPL).
(Item 27)
The preparation according to item 24, wherein the length of the degraded residual cell culture DNA is less than 500 base pairs.
(Item 28)
The preparation according to item 24, wherein the length of the degraded residual cell culture DNA is less than 200 base pairs.
(Item 29)
26. The formulation according to item 26, wherein the residual cell culture DNA is degraded by treatment with less than 0.1% β-propiolactone (BPL).
(Item 30)
The preparation according to item 24, wherein the preparation exhibits a reduced level of aggregation.
(Item 31)
A preparation containing a recombinant protein expressed in a cell culture, wherein the length of the residual functional cell culture DNA is less than 500 base pairs.
(Item 32)
The cell cultures are MDCK cells, Vero cells, and PER. The preparation according to item 24 or 31, which is selected from the group consisting of C6 cells.

FIG. 1 shows the results of PCR amplification of six named targets in the MDCK genome. One sample was diluted as described to 1: 10000 prior to PCR. FIG. 2 shows the results of PCR amplification of six named targets in the MDCK genome. One sample was diluted as described to 1: 10000 prior to PCR. FIG. 3 shows the results of PCR amplification of SINE sequences in the MDCK genome. DNA was occasionally diluted prior to PCR, as in FIGS. 1 and 2. In the lower panel, the figure is the starting amount of DNA in PCR (fg). FIG. 4 shows the effect of BPL treatment on the size of MDCK DNA. Genomic DNA was incubated with BPL at different temperatures and for different lengths of time. FIG. 5 shows similar results. The agarose gel was stained with SYBRGold. FIG. 6 shows a capillary electrophoresis of residual DNA (upper line) against a marker (lower line) having the stated size.

Detailed Description The present invention provides improved cell culture products and processes with reduced impurities. Specifically, the invention provides an improved method of degrading any residual functional cell culture DNA associated with a product made in cell culture. According to the present invention, residual functional cell culture DNA is degraded by treatment with a DNA alkylating agent, such as β-propiolactone (BPL). This process can be used to process a series of cell culture products containing vaccines and recombinant proteins.
The present invention comprises a vaccine comprising an immunogenic protein derived from a virus propagated in a cell culture, wherein the vaccine is substantially free of residual functional cell culture DNA. Furthermore, the present invention relates to a recombinant protein expressed in a cell culture, wherein the final recombinant protein preparation is substantially free of residual functional cell culture DNA.

The functionality of any residual host cell DNA can be eliminated by processing the DNA with an alkylating agent that cleaves the DNA into sufficiently small portions, thus encoding functional proteins, in the chromosomes of human recipients. It cannot be introduced into or otherwise recognized by the recipient DNA replication mechanism. The length of degraded (non-functional) residual cell culture DNA is preferably less than 1000 base pairs (eg, 1000, 800, 700, 600, 500, 400, 300, 200, 150, 100, 75, or less than 50 base pairs). Preferably, the length of the degraded residual cell culture DNA is less than 500 base pairs. More preferably, the length of the degraded residual cell culture DNA is less than 200 base pairs.

As used herein, reference to "functional DNA" or "functional RNA" indicates a nucleotide sequence that can be translated into a functional protein or introduced into a mammalian chromosome. In general, a nucleotide sequence that can be translated into a functional protein requires a promoter region, a start codon, a stop codon, and an internal coding sequence for the functional protein. When the addition of an alkylating agent causes DNA damage, many of these regions are altered or destroyed, resulting in translation being can longer processed or oligopeptides of the intended protein. It only advances to form a subunit.

"Degraded residual functional cell culture DNA" refers to functional DNA that cannot be translated into a functional protein or introduced into a mammalian chromosome. Preferably, the "degraded residual functional DNA" has a length of less than 1000 base pairs, more preferably less than 500 base pairs, even more preferably less than 250 base pairs, and most preferably less than 100 base pairs. The length of degraded residual functional DNA can be measured by standard techniques, including gel electrophoresis.

The present invention provides vaccine compositions and recombinant protein formulations that are substantially free of residual functional cell culture DNA. As used herein, a composition or formulation that is substantially free of residual functional cell culture DNA is detectable with less than 10 ng of residual DNA fragment per 0.5 ml. Point to. The size of any residual cell culture DNA can be measured by standard techniques, including capillary gel electrophoresis and nucleic acid amplification techniques.

The use of alkylating agents such as BPL in the present invention provides the additional advantage of reducing aggregation and contaminants. Vaccine formulations containing reduced aggregates may also have improved immunogenicity. The immunogenicity of a vaccine depends on the specificity of the antibody for a particular viral epitope. If the surface of the protein is bound or masked by an unwanted molecule, or is concealed by aggregation in a large macromolecule, the epitope can be less recognizable and therefore less effective in the vaccine. Can disappear. In addition, vaccine formulations containing reduced aggregates may have additional processing benefits. The purification process relies on the isolation of expected proteins such as hemagglutinin and neuraminidase in influenza vaccines. If the protein is structurally modified by the presence of aggregates or crosslinks, it cannot be recognized and can then be removed by column chromatography, filtration, or centrifugation.

Alkylating Agent Examples of the alkylating agent for use in the present invention include substances that introduce an alkyl group into a compound. Preferably, the alkylating agent is a monoalkylating agent, such as BPL. BPL is a monoalkylating agent widely used for viral inactivation in the preparation of many vaccines. The BPL reacts with various biological molecules, including nucleic acids, where it causes structural modifications by alkylation and depurination reactions. BPL is generally represented by the following structure:

インビトロにおいて、ＢＰＬは、一般的に、求核置換反応に有利な条件下で（例えば、高熱、高ＢＰＬ濃度、および非プロトン性極性溶媒において）、高濃度で存在する求核試薬と反応し、官能化プロピオン酸、例えば、７－（２－カルボキシエチル）グアニンまたは１－（２－カルボキシエチル）デオキシアデノシンが形成される（スキーム１）。Ｂｏｕｔｗｅｌｌら，Ａｎｎａｌｓ Ｎｅｗ Ｙｏｒｋ Ａｃａｄｅｍｙ ｏｆ Ｓｃｉｅｎｃｅｓ，７５１－７６４；Ｐｅｒｒｉｎら，Ｂｉｏｌｏｇｉｃａｌｓ，２３（１９９５）２０７－２１１；Ｃｈｅｎら，Ｃａｒｃｉｎｏｇｅｎｅｓｉｓ，２（２）（１９８１）７３－８０。

In vitro, BPL generally reacts with nucleophiles present at high concentrations under conditions favorable to the nucleophilic substitution reaction (eg, in high heat, high BPL concentrations, and aprotic and aprotic polar solvents). A functionalized propionic acid, such as 7- (2-carboxyethyl) guanine or 1- (2-carboxyethyl) deoxyadenosine, is formed (Scheme 1). Boutwell et al., Annals New York Academy of Sciences, 751-764; Perrin et al., Biologicals, 23 (1995) 207-211; Chen et al., Carcinogenesis, 2 (2) (1981) 73-80.

Scheme 1 (from Chen et al.):

ＤＮＡ塩基のこのような結合またはアルキル化は、塩基対置換、特に脱プリン反応、欠失、およびヌクレオシドの架橋を含む、多数の機構によって突然変異原性（ｍｕｔａｇｅｎｉｃｉｔｙ）を誘発する。ＢＰＬの高度の突然変異原性および反応性は、迅速なウイルスの死滅、および非発癌性副生成物への引き続いてのＤＮＡ分解に対応する。

Such binding or alkylation of DNA bases induces mutagenicity by a number of mechanisms, including base pair substitution, especially depurination, deletion, and nucleoside cross-linking. The high mutagenicity and reactivity of BPL corresponds to rapid viral killing and subsequent DNA degradation to non-carcinogenic by-products.

Any residual functional cell culture DNA with less than 1% BPL (eg, 1%, 0.75%, 0.5%, 0.25%, 0.2%, 0.1%, 0.075) %, 0.05%, 0.025%, 0.01%, or less than 0.005%). Preferably, the residual functional cell culture DNA is degraded by treatment with 0.1% to 0.01% BPL.

The alkylating agent is preferably added to the buffered solution and the pH of the solution is preferably maintained at 5-10. More preferably, the pH of the solution is maintained at 6-9. Even more preferably, the pH of the solution is maintained at 7-8.

In one method, the alkylating agent is added more than once. For example, a first BPL process may be performed, and then a second BPL process may be performed. There may be an alkylating agent removal step between the first and second treatments, but the alkylating agent is added for the second treatment without removing the residual alkylating agent from the first treatment. You may.

Preferably, the alkylating agent is also used as an inactivating agent for the virus used in the vaccine. The alkylating agent of the present invention is preferred as compared to traditional inactivating agents such as formaldehyde, which can crosslink proteins to other materials (including host cell DNA). Such cross-linking can lead to the formation of aggregates (eg, protein-protein aggregates, nucleotide combinations, and protein-nucleotide combinations). Since alkylating agents (eg, BPL) do not rely on such cross-linking mechanisms for virus inactivation, the use of such alkylating agents for virus inactivation causes aggregates and other impurities in vaccine products. Minimize the formation of. Such aggregates include proteins ionically or covalently attached to other proteins, proteins ionically or covalently attached to other nucleotides, and / or nucleotides ionically or covalently attached to other nucleotides. obtain.

As used herein, a reference to "aggregate" or "aggregate" refers to an individual unit or mass or collection of particles that are combined together to form a larger group or particle. Aggregation is performed before and after a possible aggregation step, or before and after application of an aggregate-disrupting method (eg, by detergent treatment) and gel electrophoresis (eg, Laemmli). It can be generally measured by quantitative measurement of the desired component by's system))), chromatography, solution turbidity, or precipitation studies and other methods well known in the art.

Treatment with alkylating agents, especially BPL, may include phases with varying temperatures. For example, a first phase at a low temperature (eg, between 2-8 ° C, eg, about 4 ° C), and a higher temperature, typically at least 10 ° C higher than the first phase (eg, 25-50 ° C). , For example at about 37 ° C.), there may be a second phase. This two-phase process is particularly useful when alkylating agents are used for both inactivation and DNA degradation. In a typical scheme, virus inactivation occurs during the cold phase and DNA degradation occurs during the hot phase. As described in more detail below, the elevated temperature may also facilitate the removal of the thermal alkylating agent.

Immunogenic Protein An immunogenic protein suitable for use in the present invention can be derived from any virus targeted by the vaccine. Immunogenic proteins include inactivated (or killed) viruses, attenuated viruses, split virus formulations, purified subunit formulations, viral proteins isolated, purified or induced from viruses, and viruses. It can be formulated as a virus-like particle (VLP).

The immunogenic protein of the invention is a viral antigen preferably comprising an epitope exposed on the surface of the virus during at least one step of its life cycle. Viral antigens are preferably stored across multiple serotypes or isolates. Viral antigens include antigens derived from one or more of the viruses described below and specific antigens identified below. The virus can be non-enveloped, or preferably enveloped. The virus is preferably an RNA virus, and more preferably an ssRNA virus. They may have a sense, or preferably an antisense genome. Their genomes can be unsegmented, or preferably segmented.

Orthomyxovirus: Viral antigens can be derived from orthomyxoviruses such as influenza A, B and C. Orthomixovirus antigens include hemagglutinin (HA), neuraminidase (NA), nuclear protein (NP), substrate protein (M1), membrane protein (M2), and one or more transcriptase components (PBl, PB2 and PA). Can be selected from one or more viral proteins, including. Preferred antigens include HA and NA.

Influenza antigens can be derived from an interpandemic (annual) influenza strain. Alternatively, the influenza antigen can be derived from a strain that has the potential to cause pandemic pandemic development (ie, an influenza strain with a new hemagglutinin compared to the hemagglutinin in currently spreading strains, or a bird subject. Influenza strains that are pathogenic in and have the potential to be transmitted horizontally in the human population, or influenza strains that are pathogenic to humans). Depending on the particular season and the nature of the antigen contained in the vaccine, influenza antigens can be derived from one or more hemagglutinin subtypes listed below: H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 or H16.

The influenza antigens of the present invention can be derived from avian influenza strains, particularly highly pathogenic avian influenza strains (HPAI). Alexander, Avian Dis (2003) 47 (3 Suppl): 976-81).

Further details of the influenza virus antigen are given below.

Paramyxoviridae virus: Viral antigens can be derived from Paramyxoviridae viruses, such as the genus Paramyxoviridae (RSV), the genus Paramyxoviridae (PIV) and the genus Paramyxoviridae (meat).

Pneumonia virus genus: Viral antigens can be derived from the pneumonia virus genus, eg, respiratory synthium virus (RSV), bovine respiratory synthium virus, mouse pneumonia virus, and citrus nasal tracheitis virus. Preferably, the genus Pneumonia virus is RSV. Pneumonia viral antigens can be selected from one or more of the following proteins, including: Surface Protein Fusion (F), Glycoprotein (G) and Small Hydrophobic protein (SH), Substrate proteins M and M2, nucleocapsid proteins N, P and L, and nonstructural proteins NS1 and NS2. Preferred pneumonia virus antigens include F, G and M. For example, J Gen Virol. 2004 Nov; 85 (Pt 11): 3229). The pneumonia virus genus antigen can also be formulated in the chimeric virus or derived from the chimeric virus. For example, a chimeric RSV / PIV virus can contain both RSV and PIV components.

Paramyxovirus genus: Viral antigens can be derived from paramyxoviruses such as paramyxovirus types 1-4 (PIV), mumps, Sendai virus, Simian virus 5, bovine paramyxoviridae virus and Newcastle disease virus. Preferably, the paramyxovirus is PIV or mumps. The paramyxovirus antigen can be selected from one or more of the following proteins: hemagglutinin-neuraminidase (HN), fusion proteins F1 and F2, nuclear protein (NP), phosphoprotein (P), large protein. (L), and substrate protein (M). Preferred paramyxovirus proteins include HN, F1 and F2. Paramyxovirus antigens can also be formulated in or derived from chimeric viruses. For example, a chimeric RSV / PIV virus can contain both RSV and PIV components. Commercially available mumps vaccines include live attenuated mumps virus (MMR) in monovalent form or in combination with measles and rubella vaccines.

Measles virus genus: Viral antigens can be derived from the measles virus genus, eg measles. The measles virus genus antigen can be selected from one or more of the following proteins: hemagglutinin (H), glycoprotein (G), fusion factor (F) large protein (L), nuclear protein (NP), polymerase phosphorus protein. (P), and substrate (M). Commercially available measles vaccines include live attenuated measles virus (MMR), typically combined with mumps and rubella.

Viruses of the Picornavirus family: Viral antigens can be derived from viruses of the Picornavirus family, such as the genus Enterovirus, the genus Rhinovirus, the genus Heparnavirus, the genus Cardiovirus and the genus Aftvirus. Antigens derived from the genus Enterovirus, such as poliovirus, are preferred.

Enterovirus genus: The virus antigens are enterovirus genus, for example, poliovirus 1, 2 or 3, coxsackie A virus types 1 to 22 and 24, coxsackie B virus types 1 to 6, echovirus (ECHO) virus) 1-9, It can be derived from types 11-27 and 29-34, as well as enterovirus 68-71. Preferably, the enterovirus genus is poliovirus. The enterovirus antigen is preferably selected from one or more of the following capsid proteins VP1, VP2, VP3 and VP4. Commercially available polio vaccines include inactivated polio vaccine (IPV) and oral poliovirus vaccine (OPV).

Heparnavirus: The viral antigen can be derived from a hepatitis virus, such as hepatitis A virus (HAV). Examples of the commercially available HAV vaccine include an inactivated HAV vaccine.

Togaviridae: Viral antigens can be derived from the Togaviridae, eg, the genus Rubivirus, the genus Alphavirus, or the genus Arterivirus. Antigens derived from the genus Rubivirus, such as rubella virus, are preferred. The togaviridae antigen can be selected from E1, E2, E3, C, NSP-1, NSPO-2, NSP-3 or NSP-4. The togaviridae antigen is preferably selected from E1, E2 or E3. Commercially available rubella vaccines include live cold-adapted viruses (MMRs), typically combined with mumps and measles vaccines.

Flavivirus genus: Viral antigens are flavivirus genus, eg, tick-borne encephalitis (TBE), dengue fever (type 1, 2, 3 or 4), yellow fever, Japanese encephalitis, western Nile encephalitis, St. Louis encephalitis, Russian spring-summer encephalitis. , Can be derived from Poissan encephalitis. Flaviviridae antigens can be selected from PrM, M, C, E, NS-1, NS-2a, NS2b, NS3, NS4a, NS4b, and NS5. The flavivirus antigen is preferably selected from PrM, M and E. Examples of the commercially available TBE vaccine include an inactivated virus vaccine.

Pestivirus: The antigen can be derived from the genus Pestivirus, eg, bovine viral diarrhea (BVDV), classical swine fever (CSFV) or border disease (Border disease) (BDV).

Hepadnavirus: Viral antigens can be derived from hepadnavirus, such as hepatitis B virus. The hepadnavirus antigen can be selected from surface antigens (L, M and S), core antigens (HBc, HBe). Examples of commercially available HBV vaccines include subunit vaccines containing the surface antigen S protein.

Hepatitis C virus: Viral antigens can be derived from hepatitis C virus (HCV). The HCV antigen is from one or more E1, E2, E1 / E2, NS345 polyproteins, NS 345-core polyproteins, cores, and / or peptides from non-structural regions (Hepatology (1991) 14:381). Can be selected.

Rhabdoviridae virus: Viral antigens can be derived from rhabdoviridae viruses such as Lyssavirus (mad dog disease virus) and Becyclovirus (VSV). The rhabdovirus antigen can be selected from glycoprotein (G), nucleoprotein (N), large protein (L), and nonstructural protein (NS). Over-the-counter rabies virus vaccines include killing viruses propagated in human diploid cells or fetal rhesus monkey lung cells.

Caliciviridae: Viral antigens can be derived from Caliciviridae, eg, nowalk viruses, as well as nowalk-like viruses, such as Hawaii virus and Snow Mountain Virus.

Coronavirus genus: Viral antigens can be derived from coronavirus, SARS, human respiratory coronavirus, avian infectious bronchitis (IBV), mouse hepatitis virus (MHV), and porcine infectious gastroenteritis virus (TGEV). The coronavirus antigen can be selected from spike (S), envelope (E), substrate (M), nucleocapsid (N), and hemagglutinin-esterase glycoprotein (HE). Preferably, the coronavirus antigen is derived from the SARS virus. The SARS virus antigen is described in WO 04/92360.

Retrovirus: Viral antigens can be derived from retroviruses such as oncoviruses, lentiviruses or spumaviruses. The oncovirus antigen can be derived from HTLV-1, HTLV-2 or HTLV-5. Lentiviral antigens can be derived from HIV-1 or HIV-2. Retroviral antigens can be selected from gag, pol, env, tax, tat, lex, rev, nef, viv, vpu, and vpr. The HIV antigen can be selected from gag (p24gag and p55gag), env (gp160 and gp41), pol, tat, nef, rev vpu, miniproteins, (preferably p55gag and gp140v deleted forms (delete)). The HIV antigen can be derived from one or more of the following strains: HIV _IIIb , HIV _SF2 , HIV _LAV , HIV _LAI , HIV _MN , HIV-1 _CM235 , HIV-1 _US4 .

Reoviridae: Viral antigens can be derived from the genus Reoviridae, such as orthovirus, rotavirus, orbivirus, or cortivirus. The Reoviridae antigen can be selected from the structural proteins λ1, λ2, λ3, μ1, μ2, σ1, σ2, or σ3, or the nonstructural proteins σNS, μNS, or σ1s. Preferred Reoviridae antigens can be derived from rotavirus. Rotavirus antigens can be selected from VP1, VP2, VP3, VP4 (or cleavage products VP5 and VP8), NSP1, VP6, NSP3, NSP2, VP7, NSP4, or NSP5. Preferred rotavirus antigens include VP4 (or cleavage products VP5 and VP8), and VP7.

Parvoviridae: Viral antigens can be derived from the genus Parvoviridae, such as parvovirus B19. The parvovirus antigen can be selected from VP1, VP-2, VP-3, NS-1 and NS-2. Preferably, the parvovirus antigen is the capsid protein VP-2.

Delta hepatitis virus (HDV): Viral antigens can be derived from HDV, especially the δ-antigen from HDV (see, eg, US Pat. No. 5,378,814).

Hepatitis E virus (HEV): Viral antigens can be derived from HEV.

Hepatitis G virus (HGV): Viral antigens can be derived from HGV.

Human herpesvirus: Viral antigens are human herpesviruses such as herpes simplex virus (HSV), varicella-zoster virus (VZV), Epstein-bar virus (EBV), cytomegalovirus (CMV), human herpesvirus 6 ( It can be derived from HHV6), human herpesvirus 7 (HHV7), and human herpesvirus 8 (HHV8). The human herpesviral antigen can be selected from the immediate early protein (α), the early protein (β), and the late protein (γ). The HSV antigen can be derived from the HSV-1 or HSV-2 strain. The HSV antigen can be selected from glycoproteins gB, gC, gD and gH, fusion proteins (gB), or antigenic escape proteins (gC, gE, or gI). The VZV antigen can be selected from core, nucleocapsid, tegument, or enveloped protein. Live attenuated VZV vaccines are commercially available. The EBV antigen can be selected from the glycoproteins of the initial antigen (EA) protein, the viral capsid antigen (VCA), and the membrane antigen (MA). The CMV antigen can be selected from capsid proteins, enveloped glycoproteins (eg, gB and gH), and segmentation proteins.

Papovavirus: The antigen can be derived from papovavirus, such as papovavirus and polyomavirus genera. As papillomaviruses, HPV serotypes 1, 2, 4, 5, 6, 8, 11, 13, 16, 18, 31, 33, 35, 39, 41, 42, 47, 51, 57, 58, 63 and 65 is mentioned. Preferably, the HPV antigen is derived from serotype 6, 11, 16 or 18. The HPV antigen can be selected from capsid proteins (L1) and (L2), or E1-E7, or a fusion thereof. The HPV antigen is preferably formulated into virus-like particles (VLPs). Examples of the polyomavirus genus virus include BK virus and JK virus. The polyomavirus antigen can be selected from VP1, VP2 or VP3.

Vaccines, 4th edition (Plotkin and Orange ed. 2004); Medical Microbiology 4th edition (Murray et al. 2002); Virology, 3rd edition (WK Joklik ed. 1988); Fundamental Virology. The viral antigens described in. Fields and DM Knipe ed. 1991) are further provided, which are considered with the compositions of the invention.

Immunogenic proteins suitable for use in the present invention can be derived from viruses that cause respiratory disease. Examples of such respiratory antigens are orthomixovirus (influenza), pneumonia virus (RSV), paramyxovirus (PIV), measles virus (measles), togavirus (waste), VZV, and corona. Examples include proteins derived from respiratory viruses such as the genus Virus (SARS). Immunogenic proteins derived from influenza virus are particularly preferred.

The compositions of the invention may comprise one or more immunogenic proteins suitable for use in pediatric subjects. Pediatric subjects are typically less than about 3 years old, or less than about 2 years old, or less than about 1 year old. The pediatric antigen can be administered multiple times over 6 months, 1, 2 or 3 years. Pediatric antigens can be derived from viruses that can target the pediatric population and / or viruses that are susceptible to infection of the pediatric population. Pediatric virus antigens include orthomixovirus (influenza), pneumonia virus (RSV), paramyxovirus (PIV and mumps), measles virus (meat), togavirus (waste), enterovirus (porio), HBV. , Coronavirus (SARS), and varicella-zoster virus (VZV), Epstein-Burvirus (EBV), and one or more derived antigens.

The compositions of the invention may comprise one or more immunogenic proteins suitable for use in elderly or immunocompromised individuals. Such individuals may need to be vaccinated more frequently at higher doses or with adjuvanted formulations to improve their immune response to the targeted antigen. Antigens that can be targeted for use in elderly or immunocompromised individuals include antigens derived from one or more of the following viruses: orthomixovirus (influenza), pneumonia virus genus (RSV), paramixo. Virus genus (PIV and mumps), measles virus genus (mellitus), togavirus (wheal), enterovirus genus (porio), HBV, coronavirus genus (SARS), varicella-belt virus (VZV), Epstein bar virus ( EBV), and cytomegalovirus (CMV).

After the virus has propagated, the alkylating agent is applied to the purified virions, eg, to the virions present in the purified cell culture, or from such purified cell cultures. Can be used for purified virions. The methods of the invention may include removing cellular material by purification followed by purification of virions from the purified cell culture (eg, by chromatography). Alkylating agents can be used for virions purified in this manner or after any further step of ultrafiltration / diafiltration. A preferred method is to use an alkylating agent against the purified supernatant of the infected cell culture, but against the virions purified from such purified supernatant (Morgeaux et al. (1993) Vaccine. 11: 82-90).

The alkylating agent is preferably used after the endotoxin removal step has been performed.

METHODS The vaccine compositions of the present invention can be prepared by isolation of immunogenic proteins and degradation of residual functional host cell DNA. Similarly, recombinant protein formulations can be prepared by isolation or purification of recombinant proteins and degradation of residual functional host cell DNA. These steps can be performed continuously or simultaneously. The step of degrading residual functional cell culture DNA is performed by the addition of an alkylating agent (eg, BPL).

Alkylating agents and any residual by-products are preferably removed prior to the final formulation of the vaccine or recombinant protein. Preferably, the vaccine composition or recombinant protein formulation contains less than 0.1% of the combined free propionic acid and BPL (eg, 0.1%, 0.05%, 0.025%, 0. 01%, 0.005%, 0.001%, or less than 0.01%). Preferably, the final vaccine composition or recombinant protein formulation contains less than 0.01% BPL.

BPL can be conveniently removed by heating and causing hydrolysis to non-toxic β-hydroxypropionic acid. The length of time required for hydrolysis depends on the total amount of BPL and the temperature. Higher temperatures provide faster hydrolysis, but temperatures should not rise high enough to damage active protein-like components. As described below, heating to about 37 ° C. for 2 to 2.5 hours is suitable for removing the BPL.

After processing the DNA, the residual DNA product can be retained in the final immunogenic or recombinant composition. However, more preferably, they are separated from the desired component, eg, from virions / proteins. Separation in this mode can partially or totally remove DNA degradation products, and preferably remove any degraded DNA that is> 200 bp. Therefore, the method of the present invention may include the step of separating DNA from virions. This separation step includes, for example, one or more ultracentrifugation, ultracentrifugation (including gradient ultracentrifugation), virus core pelleting and supernatant fractionation, chromatography (eg, ion exchange chromatography, eg, anion exchange), adsorption. Etc. may be included.

Overall, therefore, the method may include treatment with an alkylating agent to degrade the length of the residual DNA, and subsequent purification (including removal of the degraded DNA) to remove the residual DNA. ..

Cell culture The vaccine of the invention is prepared from a virus that propagates in a cell culture. Further, the present invention includes a preparation of a recombinant protein expressed in a cell culture. Mammalian cell cultures are preferred for both viral replication and recombinant protein expression.

Numerous mammalian cell lines are known in the art and include cell lines derived from: human or non-human primate (eg monkey) cells (eg WO01 / 38362, WO01 / 41814, W02 / 40665, WO2004 / 056979, and WO2005 / 080556 (all of which are incorporated herein by reference), and deposited under ECACC deposit number 96022940, PER. C6 cells), MRC-5. (ATCC CCL-171), WI-38 (ATCC CCL-75), HEK cells, HeLa cells, fetal red-tailed monkey lung cells (ATCC CL-160), human embryonic kidney cells (sheared by adenovirus type 5 DNA). Typically transformed 293 cells), Vero cells (from monkey kidneys), horses, bovines (eg MDBK cells), sheep, dogs (eg MDCK cells from canine kidneys, ATCC CCL34 MDCK (NBL2) or MDCK 33016, deposit number DSM ACC 2219 (described in WO 97/37000 and WO 97/37001), cats, and rodents (eg, hamster cells such as BHK21-F, HKCC cells, or Chinese hamster ovary (eg, BHK21-F, HKCC cells). CHO) cells); and can be obtained from a wide range of developmental stages, including, for example, adults, newborns, fetuses, embryos.

Suitable monkey cells are, for example, African green monkey cells, such as renal cells such as those in Vero cell lines. Suitable dog cells are, for example, renal cells as in the MDCK cell line. Therefore, suitable cell lines include, but are not limited to: MDCK; CHO; 293T; BHK; Vero; MRC-5; PER. C6; WI-38 etc. The use of mammalian cells means that the vaccine may not contain substances such as chicken DNA, egg proteins (eg, ovalbumin and ovomucoid), thereby reducing allergenicity.

In certain embodiments, the cells are immortalized (eg, PER. C6 cells; ECACC 96022940). In a preferred embodiment, mammalian cells are used and can be selected and / or derived from one or more of the following non-limiting cell types: fibroblasts (eg, skin, lung), endothelial cells (eg, aorta). , Coronary arteries, pulmonary arteries, vessels, cutaneous microvessels, umbilical cord), hepatocytes, keratinocytes, immune cells (eg, T cells, B cells, macrophages, NK, dendritic cells), breast cells (eg, epithelium), smooth muscle Cells (eg, vessels, aorta, coronary arteries, arteries, uterus, bronchi, neck, peri-retinal skin cells), melanocytes, nerve cells (eg, stellate cells), prostate cells (eg, epithelium, smooth muscle), kidneys or Renal cells (eg, epithelium, mesangium, proximal tubules), bone cells (eg, chondrocytes, osteotomy cells, osteoblasts), muscle cells (eg, myoblasts, skeletal muscle, smooth muscle, bronchi), Hepatocytes, retinal cells or retinal blast cells, lung cells, and stromal cells.

WO97 / 37000 and WO97 / 37001 describe the production of animal cells and cell lines that can grow in suspensions and serum-free media and are useful for the production and replication of viruses (particularly influenza viruses). Further details are given in WO 03/023021 and WO 03/023025.

Preferred mammalian cell lines for propagating influenza virus include: MDCK cells from Madin Davy canine kidneys; Vero cells from African green monkey (Cercopisecus aethios) kidneys; or human germ retinal blast cells. , PER. C6 cells. These cell lines are widely available, for example, from the American Type Cell Culture (ATCC) collection, from the Coriell Cell Repositories, or from the European Collection of Cell Cultures (ECACC). For example, the ATCC supplies a variety of different Vero cells with catalog numbers CCL-81, CCL-81.2, CRL-1586 and CRL-1587, and it supplies MDCK cells with catalog number CCL-34. PER. C6 is available from ECACC with deposit number 96022940.

The most preferred cell line for growing influenza virus is the MDCK cell line. The original MDCK cell line is available from the ATCC as CCL-34, but derivatives of this cell line can also be used. For example, WO 97/37000 discloses an MDCK cell line adapted for growth in suspension culture (“MDCK 33016” deposited as DSM ACC 2219). Similarly, EP-A-1260581 (WO 01/64846) discloses an MDCK-derived cell line that grows in suspensions in serum-free culture (Deposited as FERM BP-7449, "B-702". ). WO2006 / 071563 includes "MDCK-S" (ATCC PTA-6500), "MDCK-SF101" (ATCC PTA-6501), "MDCK-SF102" (ATCC PTA-6502) and "MDCK-SF103" (PTA-6503). ), Non-tumorogenic MDCK cells are disclosed. WO2005 / 113758 discloses an MDCK cell line that is highly susceptible to infection, including "MDCK.5F1" cells (ATCC CRL-12042). Any of these MDCK cell lines can be used.

Operations on MDCK cell cultures and adherent cultures in suspension are described in WO97 / 37000, WO97 / 37001, WO03 / 023021, and WO03 / 023025. In particular, WO 03/023021 and WO 03/023025 describe MDCK suspended cells in experimental and commercial scale cell culture volumes in serum-free media, chemically defined media, and protein-free media. ing. Each reference is incorporated herein by reference in its entirety.

As an alternative to mammalian sources, cell lines for use in the present invention can be derived from avian sources such as chickens, ducks, geese, quails or pheasants. Avian cell lines can be derived from a variety of developmental stages, including embryos, juveniles and adults. Preferably, the cell line is an embryonic cell, such as an embryonic fibroblast, a germ cell, or an individual organ (including neurons, brain, retina, kidney, liver, heart, muscle), or extraembryonic tissue that protects the embryo. Derived from the membrane. Examples of avian cell lines include avian embryonic stem cells (WO01 / 85938 and WO03 / 076601) and duck retinal cells (WO2005 / 042728). Suitable avian embryonic stem cells include EBx cell lines derived from chicken embryonic stem cells, EB45, EB14, and EB14-074 (WO2006 / 108846). Chicken embryo fibroblasts (CEF) can also be used. These avian cells are particularly suitable for propagating influenza virus.

Insect cell expression systems, such as baculovirus recombinant expression systems, are known to those of skill in the art and, for example, Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). Materials and methods for the baculovirus / inserted cell expression system are commercially available, especially in kit form, manufactured by Invitrogen, San Diego CA. Insect cells for use with the baculovirus expression vector include, among others, Aedes aegipti, Autographa californica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, and Tri.

Recombinant expression of proteins can also occur in bacterial hosts such as Escherichia coli, Bacillus subtilis, and Streptococcus species. Suitable yeast host for recombinant expression of a protein, Saccharomyces cerevisiae, Candida albicans, Candida maltosa, Hansenual polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris, Schizosaccharomyces pombe, and Yarrowia lipolytica and the like.

Culture conditions for the cell types are well described in various publications, or culture media, supplements and conditions are described, for example, in the Catalog of Cambrex Bioproducts (East Rutherford, NJ) and further literature. As such, it can be purchased commercially.

In certain embodiments, the host cells used in the methods described herein are cultured in serum-free and / or protein-free medium. Medium is referred to as serum-free medium in the context of the present specification in the absence of serum-derived additives of human or animal origin. Protein-free is a protein such as trypsin or other proteases that may be required for viral growth, although cellular multiplication occurs with the elimination of proteins, growth factors, other protein additives and non-serum proteins. It is understood to mean a culture that can be included as needed. Cells proliferating in such cultures can themselves contain proteins naturally.

Known serum-free media include Iskove medium, Ultra-CHO medium (BioWhittaker) or EX-CELL (JRH Bioscience). Typical serum-containing media include Eagle's Basic Medium (BME) or Minimal Essential Medium (MEM) (Eagle, Science, 130, 432 (1959)) or Dulbecco's Modified Eagle's Medium (DMEM or EDM). Usually used with up to 10% fetal bovine serum or similar additives. If desired, Minimal Essential Medium (MEM) (Eagle, Science, 130, 432 (1959)) or Dulbecco's Modified Eagle's Medium (DMEM or EDM) can be used without serum-containing supplements. Protein-free media such as PF-CHO (JHR Bioscience), chemically defined media such as ProCHO ^4CDM (BioWhittaker) or SMIF 7 (Gibco / BRL Life Technologies), and Primactone, Peptide (International) mitogen peptides or lactoalbumin hydrolysates (Gibco and other manufacturers) are also well known in the art. Medium additives based on plant hydrolysates have the special advantage that contamination with viruses, mycoplasmas or unknown infectious factors can be eliminated.

Cell culture conditions (temperature, cell density, pH values, etc.) are very widely variable due to the suitability of the cell lines used in accordance with the present invention, and are specific viral growth conditions or recombinant expression details. Can be adapted to requirements.

The cells can grow in various ways, eg, in suspension, in adherent culture, on microcarriers.

Cells can grow below 37 ° C (eg, 30-36 ° C) during viral replication (WO97 / 37001).

Methods for growing a virus in a cultured cell are generally desired for the step of infecting the cultured cells with the cultured strain, eg, for virus growth, as measured by virus titer or antigen expression. Includes a step of culturing infected cells and a step of recovering the propagated virus for a period of time (eg, 24-168 hours after inoculation). The cultured cells have a virus (measured by PFU or TCID ₅₀ ) to cell ratio of 1: 500 to 1: 1, preferably 1: 100 to 1: 5, more preferably 1: 50 to 1:10. Can be inoculated with. The virus can be added to a suspension of cells or applied to a monolayer of cells, and the virus can be applied for at least 60 minutes but usually less than 300 minutes, preferably 90-240 minutes, 25 ° C-40. It is adsorbed onto cells at ° C., preferably 28 ° C. to 37 ° C.

The infected cell culture (eg, monolayer) can be removed by freeze-thaw or enzymatic action to increase the viral content of the harvested culture supernatant. The harvested fluid is then inactivated or cryopreserved. Cultured cells can be infected with a multiplicity of infection (“moi.”) Of about 0.0001-10, preferably 0.002-5, more preferably 0.001-2. Even more preferably, the cells are about 0.01 m. o. i. Infected with. Infected cells can be harvested 30-60 hours after infection. Preferably, the cells are harvested 34-48 hours after infection. Even more preferably, the cells are harvested 38-40 hours after infection. Proteases (typically trypsin) are generally added during cell culture to allow viral shedding, and the protease can be added at any suitable stage during culture.

Vaccine compositions of the invention are generally in sub-virion form, eg, in the form of a split virus (where the viral lipid envelope is lysed or destroyed), or one or more purifications. It is formulated in the form of the viral protein. The vaccine composition contains an amount of antigen sufficient to elicit an immunological response in the patient.

Methods for splitting viruses such as influenza virus are well known in the art and see, for example, WO02 / 28422, WO02 / 067983, WO02 / 07433, WO01 / 21151 and the like. Virus splitting is by disrupting or fragmenting the entire virus, whether infectious (wild-type or attenuated) or non-infectious (eg, inactivated), with a destructive concentration of splitting agent. Will be done. Splitting agents generally include agents capable of breaking and dissolving lipid membranes, typically having a hydrophobic tail bound to a hydrophilic head. The most preferred splitting agent is cetyltrimethylammonium bromide (CTAB). Further details of the division are given below in the context of influenza virus.

Destruction results in complete or partial solubilization of viral proteins, altering viral integrity. Preferred dividers are non-ionic and ionic (eg, cationic) surfactants such as alkyl glycosides, alkylthioglycosides, acyl sugars, sulfobetaines, betaines, polyoxyethylene alkyl ethers, N, N. -Dialkyl-glucamide, Hecameg, alkylphenoxy-polyethoxyethanol, quaternary ammonium compounds, sarcosyl, CTAB (cetyltrimethylammonium bromide), tri-N-butyl phosphate, Cetavlon, myristyltrimethylammonium salt, lipofectin, lipofectamine, and DOT-MA, octyl or nonylphenoxypolyoxyethanol (eg, Triton surfactant, eg Triton X-100 or Triton N101), polyoxyethylene sorbitan ester (Tween surfactant), polyoxyethylene ether, polyoxyethylene ester. And so on.

Methods of purifying individual proteins from viruses are well known and include, for example, filtration, chromatography, centrifugation and hollow fiber elution. In one embodiment, the protein is purified by an ion exchange resin.

As a further alternative, the vaccine may include a total virus, eg, a live attenuated total virus, or preferably an inactivated total virus. Methods of inactivating or killing viruses to destroy their ability to infect mammalian cells are known in the art. Such methods include both chemical and physical means. Chemical means for inactivating the virus include treatment with one or more of the following agents in an effective amount: detergent, formaldehyde, formalin, BPL, or UV light. Further chemical means for inactivation include treatment with methylene blue, psoralen, carboxyfullerene (C60) or any combination thereof. Other methods of virus inactivation, such as binary ethylamine, acetylethyleneimine, or gamma-ray irradiation, are known in the art. Preferably, the virus is inactivated by BPL.

Pharmaceutical Compositions The compositions of the present invention are pharmaceutically acceptable. They usually contain components in addition to the antigen, eg, they typically contain one or more pharmaceutical carriers and / or excipients. A complete discussion of such components is available in Remington: The Science and Practice of Pharmacy (Gennaro, 2000; 20th Edition, ISBN: 0683306472).

The composition is generally in an aqueous form.

The composition may include one or more preservatives, such as thiomersal or 2-phenoxyethanol. However, the vaccine should not contain substantially any mercury material (ie, less than 5 μg / ml) and is preferably thiomethal-free, for example (Banzahoff (2000) Immunology Letters 71: 91-96; WO 02/097072). .. Vaccines that do not contain mercury are more preferred. Vaccines that do not contain preservatives are particularly preferred.

For example, it is preferable to include a physiological salt (for example, a sodium salt) in order to control the tonicity. Sodium chloride (NaCl) is preferred, which can be present at 1-20 mg / ml. Other salts that may be present include potassium chloride, potassium dihydrogen phosphate, disodium phosphate dehydrate, magnesium chloride, calcium chloride and the like.

The composition generally has a weight osmolal concentration of 200 mOsm / kg to 400 mOsm / kg, preferably 240 to 360 mOsm / kg, and more preferably falls within the range of 290 to 310 mOsm / kg.

The composition may contain one or more buffers. Typical buffers include:
Phosphate buffer; Tris buffer; Borate buffer; Succinic acid buffer; Histidine buffer (particularly containing aluminum hydroxide adjuvant); or Citrate buffer. The buffer is typically contained in the 5-20 mM range.

The pH of the composition is generally 5.0 to 8.1, and more typically 6.0 to 8.0, such as 6.5 to 7.5. Therefore, the method of the present invention may include adjusting the pH of a bulk vaccine prior to packaging.

The composition is preferably sterile. The composition is preferably non-pyrogenic and contains, for example, <1 EU (endotoxin unit, standard measurement) per dose, and preferably <0.1 EU per dose. The composition preferably contains no gluten.

The compositions of the present invention are detergents such as polyoxyethylene sorbitan ester surfactants (known as "Tweens"), octoxinol (eg, octoxinol-9 (Triton X-100) or t-octylphenoxypoly). It may contain ethoxyethanol), cetyltrimethylammonium bromide (“CTAB”), or sodium deoxycholate. Detergent can only be present in trace amounts.

The composition may include material for single immunization or may include material for multiple immunization (ie, "multi-dose" kit). The inclusion of preservatives is useful in high dose arrangements. As an alternative (or in addition to) the inclusion of preservatives in the multidose composition, the composition can be included in a container with an aseptic adapter for the removal of the material.

Vaccines are typically given in dosages of about 0.5 ml, but half doses (ie about 0.25 ml) can be given to children.

The compositions and kits are preferably stored at 2 ° C to 8 ° C. They should not be frozen. They should ideally be kept out of direct sunlight.

Therapeutic methods and administration of vaccines The compositions of the invention are suitable for administration to animal (and especially human) patients, and the invention comprises the step of administering the composition of the invention to a patient. Provided is a method of inducing an immune response in a patient.

The invention also provides the kit or composition of the invention for use as a pharmaceutical.

The invention also provides the use of immunogenic proteins derived from viruses propagated in cell cultures in the manufacture of pharmaceuticals to elicit an immune response in a patient, wherein the vaccine remains functional. Substantially free of cell culture DNA.

Immune responses evoked by these methods and uses generally include antibody responses, preferably protective antibody responses. Methods for assessing antibody response, neutralization capacity and protection after viral vaccination are well known in the art. For example, for influenza virus, human studies have shown that antibody titers to HA are associated with protection (about 30-40 serum sample hemagglutination inhibitory titers provide about 50% protection from infection by allogeneic viruses. Provided) [Potter & Oxford (1979) Br Med Bull 35: 69-75].

The compositions of the invention can be administered in a variety of fashions. The most preferred route of immunization is by intramuscular injection (eg, into the arm or leg), while other available routes are subcutaneous injection, intranasal, oral, intradermal, transcutaneous, transdermal. Examples include skin (transdermal) and the like.

Vaccines prepared according to the present invention can be used to treat both children and adults. Patients can be less than 1 year old, 1-5 years old, 5-15 years old, 15-55 years old, or at least 55 years old. Patients are elderly (eg, ≧ 50, ≧ 60, and preferably ≧ 65), young (eg, ≦ 5), inpatients, health care workers, military and military personnel, pregnant women, chronic. Patients with illness, immunocompromised patients, patients who took antiviral compounds (eg, oseltamivir or zanamivir compounds for influenza; see below) during the 7 days prior to receiving the vaccine, those with egg allergies, abroad It may be a traveler. However, vaccines are not suitable only for these groups and may be used more commonly in populations.

Treatment can be by a single dose schedule or a multiple dose schedule. Multiple doses can be used in a primary and / or booster immune schedule. In a multi-dose schedule, different doses may be given by the same or different routes, such as parenteral and transmucosal boosts, transmucosal primes and parenteral boosts, and the like. Two or more doses (typically two doses) are particularly useful in immunologically naive patients. Multiple doses are typically at least 1 week apart (eg, about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, about 16 weeks, etc.). Be administered.

Vaccines produced according to the invention are substantially simultaneous with other vaccines (eg, during the same medical consultation or visit to a medical professional or vaccination center), eg, bacterial vaccines such as diphtheria vaccines, tetanus. Vaccines, pertussis vaccines, DTP vaccines, combined influenza b-type vaccines, meningococcal conjugate vaccines (eg, tetravalent AC-W135-Y vaccines), pneumonia-bound vaccines It can be administered to a patient substantially simultaneously with (pneumococcal vaccine vaccine) and the like.

Similarly, the vaccines of the invention can be administered to a patient substantially simultaneously (eg, during the same medical consultation or visit with a medical professional) with an antiviral compound effective against the virus in the vaccine. For example, if the vaccine is an influenza vaccine, the compound can be a neuraminidase inhibitor (eg, oseltamivir and / or zanamivir). These antiviral agents include (3R, 4R, 5S) -4-acetylamino-5-amino-3 (eg, ethyl esters) and salts thereof (eg, phosphates). 1-Ethylpropoxy) -1-cyclohexane-1-carboxylic acid, or 5- (acetylamino) -4-[(aminoiminomethyl) -amino] -2,6-anhydro-3,4,5-trideoxy-D -Glycero-D-galactonon-2-enonic acid can be mentioned. A preferred antiviral agent effective against influenza is (3R, 4R, 5S) -4-acetylamino-5-amino-3 (1-ethylpropoxy) -1, also known as oseltamivir phosphate (TAMIFLU ^TM ). -Cyclohexene-1-carboxylic acid, ethyl ester, phosphate (1: 1).

Host cell DNA
Measurement of residual host cell DNA is within the normal capacity of one of ordinary skill in the art. The total amount of residual DNA in the composition of the present invention is preferably less than 20 ng / ml, for example, ≤10 ng / ml, ≤5 ng / ml, ≤1 ng / ml, ≤100 pg / ml, ≤10 pg / ml and the like. .. As mentioned above, substantially all of this DNA is preferably less than 500 base pairs in length.

The assay used to measure DNA is typically a certified assay (Guide for Veterinary: Bioanalytic Measurement. U.S. Department of Health and Human Division Operation Assay (CDER) Center for Veterinary Medicine (CVM). May 2001; Lundblad (2001) Biotechnology and Applied Bioanalysis
34: 195-197). The performance characteristics of the certified assay can be described in mathematical and quantifiable terms, and its possible source of error has been identified. Assays were generally tested for features such as accuracy, accuracy, and specificity. Once the assay has been calibrated and tested (eg, against a known standard amount of host cell DNA), quantitative DNA measurements can be performed as prescribed. Three principle techniques for DNA quantification can be used: hybridization methods, eg Southern blots or slot blots (Ji et al. (2002) Biotechniques. 32: 1162-7); immunological determination methods, eg. , Threshold ^TM system (Brigs (1991) J Parenter Sci Technol. 45: 7-12 .; and quantitative PCR (Lahijani et al. (1998) Hum Gene Ther. 9: 1173-80). All of these methods are well suited to those skilled in the art. As is known, the exact characteristics of each method may depend on the host cell in question, eg, the choice of probe for hybridization, the choice of primer and / or probe for amplification, etc. Threeshold from Molecular Devices. The ^TM system is a quantitative assay for total DNA at the picogram level and has been used to monitor the level of contaminated DNA in biopharmaceuticals (Briggs (1991), see above). Includes non-sequence-specific formation of reaction complexes between biotinylated ssDNA-binding proteins, urease-conjugated anti-ssDNA antibodies, and DNA. All assay components are complete total DNA available from said manufacturer. Included in the assay kit. Various commercial manufacturers provide quantitative PCR assays for detecting residual host cell DNA, such as ApTec ^TM Laboratory Services, BioReliance ^TM , Althea Technologies, etc. for human virus vaccines. A comparison of total DNA Thrashold ^TM systems and chemical luminescence hybridization assays for measuring host cell DNA contamination can be found in Laketeff et al. (2001).
Biologicals. It can be seen at 29: 123-32.

These various analytical methods can also be used to measure the length of residual host cell DNA. As mentioned above, the average length of residual host cell DNA is preferably less than 500 base pairs, or even less than 200 base pairs, after treatment with an alkylating agent.

For canine cells (especially MDCK cells), genomic analysis reveals 13 coding sequences <500 bp long, 3 sequences <200 bp, and 1 sequence <100 bp. Therefore, fragmentation of DNA into <200 bp removes virtually all coding sequences, and any fragment is about the length of three genes (ie: 81 bp secretin; 108 bp PYY; and 135 bp). It is highly unlikely that it will actually correspond to one of (osteocalcin).

Adjuvants The compositions of the invention may include an adjuvant that may function to enhance the immune response (humoral and / or cellular) elicited in the patient receiving the composition. The use of adjuvants with viral vaccines is well known, for example, in hepatitis vaccines, polio vaccines and the like. The FLUAD ^TM influenza vaccine contains an oil-in-water emulsion adjuvant.

The adjuvants that can be used with the present invention include, but are not limited to, aluminum salts, immunostimulatory oligonucleotides, saponins, Lipid A analogs (eg, 3dMPL), and oil-in-water emulsions. These and other adjuvants include Vaccine Design (Vaccine Design: The Subunit and Adjuvant Approach, Plenum Press 1995, ISBN 0-306-44867-X) and O'Hagan (Vaccine).
It is disclosed in more detail in Adjuvants: Preparation Methods and Research Protocols, volume 42 of Methods in Molecular Medicine series, ISBN: 1-59259-083-7).

An adjuvant known as aluminum hydroxide and aluminum phosphate can be used. These names are customary, but neither is an exact description of the actual compound present, so they are used only for convenience (see, eg, Chapter 9 of Powerell & Newman). The present invention may use either "hydroxide" or "phosphate" adjuvants commonly used as adjuvants. Adsorption to these salts is preferred.

Immunostimulatory oligonucleotides with adjuvant activity are well known. They are CpG motifs (dinucleotide sequences containing unmethylated cytosine linked by guanosine to phosphate), TpG motifs, oligo-dT sequences, oligo-dC sequences, double-stranded RNA, parindrome sequences, poly. It may contain (dG) sequences and the like. Immunostimulatory oligonucleotides typically contain at least 20 nucleotides and may contain less than 100 nucleotides.

Saponins (Chapter 22 of Powerell & Newman) are a heterologous group of sterol and triterpenoid glycosides found in the bark, leaves, stems, roots and even flowers of a wide range of plant species. Saponins derived from the bark of the Quillaja saponaria tree (Molina tree) have been widely studied as an adjuvant. Examples of the saponin adjuvant preparation include purified preparations such as QS21 and liquid preparations such as ISCOM. The saponin composition has been purified using HPLC and RP-HPLC, and certain purified fractions (QS7, QS17, QS18, QS21, QH-A, QH-B) using these techniques. And QH-C) were identified. Preferably, the saponin is QS21. A method of manufacturing QS21 is disclosed in US Pat. No. 5,575,540. Saponin preparations may also contain sterols, such as cholesterol (WO96 / 33739). The combination of saponin and cholesterol can be used to form unique particles called immunostimulatory complexes (ISCOMs), ISCOMs, which also typically contain phospholipids.

3dMPL (also known as 3-de-O-acylated monophosphoryl lipid A or 3-O-desacyl-4'-monophosphoryl lipid A) deaculates the 3-position of the reducing terminal glucosamine in monophosphoryl lipid A. It is an adjuvant. 3dMPL is prepared from a heptoseless mutant of Salmonella minnesota and is chemically similar to Lipid A, but lacks an acid-labile phosphoryl group and a base-unstable acyl group. The 3dMPLs can take the form of a mixture of related molecules that differ by their acylation (eg, have 3, 4, 5 or 6 acyl chains that can be of different lengths).

Various oil-in-water emulsions with adjuvant activity are known. They typically contain at least one oil and at least one surfactant, which are biodegradable (metabolic) and biocompatible. The oil droplets in the emulsion generally have a submicron diameter and these small sizes are achieved with a microfluidizer to provide a stable emulsion. Droplets having a size of less than 220 nm are preferred as they can be subjected to filtration sterilization.

Specific oil-in-water emulsion adjuvants useful in the present invention include, but are not limited to:
● Submicron emulsions of squalene, Tween 80, and Span 85. The composition of the emulsion by volume can be about 5% squalene, about 0.5% polysorbate 80, and about 0.5% Span 85. In terms of weight, these ratios are 4.3% squalene, 0.5% polysorbate 80, and 0.48% Span 85. This adjuvant is known as "MF59", as described in more detail in Chapter 10 of Powerell & Newman and Chapter 12 of O'Hagan. The MF59 emulsion preferably contains citrate ions, such as 10 mM sodium citrate buffer.

● Emulsions of squalene, tocopherols, and Tween 80. The emulsion may contain phosphate buffered saline. It may also contain Span 85 (eg at 1%) and / or lecithin. These emulsions can have 2-10% squalene, 2-10% tocopherols, and 0.3-3% Tween 80, and the weight ratio of squalene: tocopherols is preferably ≦ 1, because This is to provide a more stable emulsion. Squalene and Tween 80 can be present in a volume ratio of about 5: 2. One such emulsion is Tween in PBS
It can be made by dissolving 80 to provide a 2% solution, then mixing 90 ml of this solution with a mixture of (5 g DL-α-tocopherol and 5 ml squalene), and then microfluiding the mixture. The resulting emulsion may have submicron oil droplets having an average diameter of, for example, 100-250 nm, preferably about 180 nm.

● Emulsions of squalene, tocopherols, and Triton detergents (eg, Triton X-100). The emulsion may also contain 3d-MPL. The emulsion may contain a phosphate buffer.

● Emulsion containing polysorbate (eg, polysorbate 80), Triton detergent (eg, Triton X-100) and tocopherol (eg, α-tocopherol succinate). The emulsion may contain these three components in a mass ratio of about 75: 11: 10 (eg, 750 μg / ml polysorbate 80, 110 μg / ml Triton X-100, and 100 μg / ml α-tocopherol succinate). These concentrations should include any contribution of these components from the antigen. The emulsion may also contain squalene. The emulsion may also contain 3d-MPL. The aqueous phase may contain phosphate buffer.

Influenza vaccine The present invention is particularly suitable for preparing influenza virus vaccines. Various forms of influenza virus vaccines are currently available; see, for example, Chapters 17 and 18 of Plotkin & Orenstein (Vaccines, 4th Edition, 2004, ISBN: 0-7216-9688-0). They are generally based on either live or inactivated viruses. Inactivated vaccines can be based on whole virions, "divided" virions, or purified surface antigens, including hemagglutinin. Influenza antigens can also be presented in the form of virosome (nucleic acid-free virus-like liposome particles). Antigens purified from recombinant hosts (eg, in insect cell lines using baculovirus vectors) can also be used.

Chemical means for inactivating the virus include treatment with one or more of the following agents in an effective amount: detergent, formaldehyde, β-propiolactone, methylene blue, psoralen, carboxyfullerene (C60), Binary ethylamine, acetylethyleneimine, or a combination thereof. Non-chemical methods of virus inactivation, such as UV light or gamma ray irradiation, are known in the art.

Billions can be harvested from virus-containing fluids by a variety of methods. For example, the purification process may include zone centrifugation using a linear sucrose gradient solution containing a detergent that destroys virions. The antigen can then be purified by diafiltration after any dilution.

The split virions include a detergent (eg, ethyl ether, polysorbate 80, deoxycholate, tri-N-butyl phosphate, Triton X-100, Triton N101, odor), including a "Tween-ether" split process. It is obtained by treating with (cetyltrimethylammonium bromide, etc.) to prepare a subvirion preparation. Methods for splitting influenza viruses are well known in the art, such as WO02 / 28422, WO02 / 067983, WO02 / 07433, WO01 / 21151, WO02 / 097072, WO2005 / 113756. Separation of a virus is typically done by disrupting or fragmenting the entire virus, whether infectious or non-infectious, with a disruptive concentration of disrupting agent. Destruction results in complete or partial solubilization of viral proteins, altering viral integrity. Preferred dividers are non-ionic and ionic (eg, cationic) surfactants such as alkyl glycosides, alkylthioglycosides, acyl sugars, sulfobetaines, betaines, polyoxyethylene alkyl ethers, N, N-dialkyl-glucamides. , Hecameg, alkylphenoxy-polyethoxyethanol, quaternary ammonium compounds, sarcosyl, CTAB (cetyltrimethylammonium bromide), tri-N-butyl phosphate, Cetavlon, myristyltrimethylammonium salt, lipofectin, lipofectamine, and DOT-MA, Octyl or nonylphenoxypolyoxyethanol (for example, Triton surfactant, for example, Triton X-100 or Triton N101), polyoxyethylene sorbitan ester (Tween surfactant), polyoxyethylene ether, polyoxyethylene ester and the like. One useful splitting procedure uses the continuous effect of sodium deoxycholate and formaldehyde, and splitting can occur during initial virion purification (eg, in a sucrose density gradient solution). Therefore, the splitting process involves purification of virion-containing materials (to remove non-virion materials), concentration of harvested virions (eg, using adsorption methods such as CaHPO ₄ adsorption), and total virions from non-virion materials. Separation, density gradient division of virions using a dividing agent in the centrifugation step (eg, using a sucrose gradient containing a dividing agent such as sodium deoxycholate), and then removal of unwanted material. It may include filtration (eg, extrafiltration). The split virions can conveniently be resuspended in sodium phosphate buffered isotonic sodium chloride solution.

Purified surface antigen vaccines also contain influenza surface antigen hemagglutinin, and typically neuraminidase. The process for preparing these proteins in purified form is well known in the art.

Influenza virus strains for use in vaccines vary from season to season. In the current inter-pandemic period, the vaccine typically comprises two influenza A strains (H1N1 and H3N2) and one influenza B strain, and a trivalent vaccine is typical. The invention is also a pandemic strain (ie, a strain in which the vaccine recipient and the general human population are immunologically naive, such as H2, H5, H7 or H9 subtype strains (particularly those of influenza A virus). ) Derived HA can be used, and the influenza vaccine for a pandemic strain may be monovalent or may be based on a conventional trivalent vaccine supplemented by a pandemic strain. However, depending on the season and the nature of the antigen contained in the vaccine, the present invention may be one or more influenza A virus hemagglutinin subtypes H1, H2, H3, H4, H5, H6, H7, H8, H9, It may be protected from H10, H11, H12, H13, H14, H15 or H16.

The compositions of the present invention are particularly useful for immunizing a pandemic strain, as is suitable for immunizing an interpandemic strain. The characteristics of the influenza strain that give it the potential to cause a pandemic outbreak are as follows: (a) It is a new hemagglutinin compared to the hemagglutinin in human strains that are currently widespread, ie humans over 10 years. It was not apparent in the population (eg, H2) or was never previously seen in the human population (eg, was commonly found only in the avian population, H5, H6 or H9), resulting in humans. The population contains those that are immunologically naive to the hemagglutinin of the strain; (b) it can be transmitted horizontally in the human population; and (c) it is pathogenic to humans. be. Viruses with the H5 hemagglutinin type are preferred for immunizing against pandemic influenza such as the H5N1 strain. Other potential strains include H5N3, H9N2, H2N2, H7N1 and H7N7, as well as any other new potentially pandemic strains. Within the H5 subtype, the virus can enter HA Clade 1, HA Clade 1', HA Clade 2 or HA Clade 3 (World Health Organization (2005) Emerging Infectious Diseases 11 (10): 1515-21), Clade 1. And 3 are particularly relevant. Other strains in which the antigen may be usefully included in the composition are strains that are resistant to antiviral therapy (eg, resistant to oseltamivir and / or zanamivir), including resistant pandemic strains.

The compositions of the invention may comprise antigens from one or more (eg, 1, 2, 3, 4 or more) influenza virus strains, including influenza A virus and / or influenza B virus. A trivalent vaccine comprising antigens from two influenza A virus strains and one influenza B virus strain is preferred.

The influenza virus can be a reassortant strain and may have been obtained by reverse genetics techniques. Thus, influenza A virus may contain one or more RNA segments from the A / PR / 8/34 virus (typically six segments from A / PR / 8/34, HA and N segments are vaccine strains. Origin, ie 6: 2 reassortment). It may also contain one or more RNA segments derived from the A / WSN / 33 virus or from any other viral strain useful for making reassorted viruses for vaccine preparation. Typically, the invention protects against a human-to-human infectious strain, and therefore the genome of the strain usually comprises at least one RNA segment derived from a mammalian (eg, human) influenza virus. It may include NS segments derived from the avian influenza virus.

HA is the main immunogen in current inactivated influenza vaccines, and vaccine doses are standardized by reference to HA levels, typically measured by SRID. Vaccines present typically contain about 15 μg HA per strain, but lower doses can be used, for example, for children, or in pandemic situations, or when using an adjuvant. As well as higher doses (eg, 3x or 9x doses), divided doses such as 1/2 (ie, 7.5 μg HA per strain), 1/4 and 1/8 have been used. However, the vaccine is 0.1 to 150 μg of HA per influenza strain, preferably 0.1 to 50 μg, such as 0.1 to 20 μg, 0.1 to 15 μg, 0.1 to 10 μg, 0.1 to 7 It may contain .5 μg, 0.5-5 μg, etc. Specific doses include, for example, about 15, about 10, about 7.5, about 5, about 3.8, about 1.9, about 1.5, etc. per strain.

For live vaccines, dosing is measured by median tissue culture infection dose (TCID ₅₀ ) rather than HA content, and 10 ⁶ to 10 ⁸ per strain (preferably 10 ^6.5 ). The TCID ₅₀ of ~ ^107.5 ) is typical.

The HA used in the present invention may be the natural HA found in the virus or may be modified. For example, modifying HA to remove determinants that make the virus highly pathogenic in avian species (eg, the hyper-basic region around the cleavage site between HA1 and HA2). Is known.

After treating the virion-containing composition (eg, with BPL) to degrade the host cell DNA, the degradation products are preferably removed from the virion, for example by anion exchange chromatography. Once the influenza virus has been purified for a particular strain, it can be mixed with viruses from other strains, for example to make the trivalent vaccine described above. Rather than mixing the virus and degrading the DNA from the multivalent mixture, it is preferred to treat each strain separately and mix the monovalent bulk to obtain the final multivalent mixture.

The general term "comprising" includes "inclusion" and "consisting", for example, a composition that "contains" X may consist of X alone or is additional. For example, X + Y may be included.

The term "substantially" does not exclude "completely", for example, a composition "substantially free" of Y may be completely free of Y. If desired, the term "substantially" may be omitted from the definition of the present invention.

The term "about" in relation to the number x means, for example, x + 10%.

By the terms "isolated" and "purified", at least 50% pure, more preferably 60% pure (as with most divided vaccines), more preferably 70% pure, or It means 80% pure, or 90% pure, or 95% pure, or greater than 99% pure.

Unless otherwise stated, a process comprising mixing two or more components does not require any particular mixing sequence. Therefore, the components can be mixed in any order. If three components are present, the two components can be mixed with each other, and then the mixture can be mixed with a third component and the like.

When animal (and especially bovine) materials are used in cell culture, they are obtained from sources that are free of infectious spongiform encephalopathy (TSE), and particularly free of bovine spongiform encephalopathy (BSE). Should be. Overall, it is preferred to culture the cells in the complete absence of animal-derived material.

If the compound is administered to the body as part of the composition, the compound may be replaced by a suitable prodrug instead.

If the cellular substrate is used for reassortment or reverse genetic procedures, it may be, for example, Ph.
Eur is preferably licensed for use in the production of human vaccines, as in General Chapter 5.2.3.

Embodiments for carrying out the invention Influenza virus (A / New Caledonia / 20/99 (H1N1), A / Panama / 2007/99 (H3N2); B / Jiangsu / 10/2003; A / Wyoming / 3 / 2003 (H3N2)) was grown in MDCK cells during suspension culture according to the teachings of WO97 / 37000, WO03 / 23025 and WO04 / 92360. The final culture medium was purified to give virions, which were then subjected to chromatography and ultrafiltration / diafiltration. The virions in the resulting material were inactivated with β-propiolactone (final concentration 0.05% v / v; incubated at 2-8 ° C for 16-20 hours, and then at 37 ° C. Hydrolyzed by incubation for 2 to 2.5 hours). The virion was then divided using CTAB and various further treatment steps yielded the final monovalent bulk vaccine containing the purified surface antigen.

MDCK DNA was characterized in three stages of the manufacturing process and its quantity, size, and completeness were evaluated: (A) after the extrafiltration / difiltration step; (B) after β-propiolactone treatment. ; And (C) in the final monovalent bulk. Capillary gel electrophoresis and nucleic acid amplification were used to study the size, completeness and biological activity of any residual genomic DNA.

Size Measurements As mentioned above, the size of residual host cell DNA was analyzed by capillary gel electrophoresis in steps (A), (B) and (C). Analysis was performed on 5 distinct virus cultures.

500 μl samples are removed at these three time points and treated with 10 μl Proteinase K at 56 ° C. for 16-22 hours, followed by total DNA extraction with a DNA extraction kit (Wako Chemicals) according to the manufacturer's instructions. Was done. The DNA was resuspended in 500 μl ultrapure water at a constant temperature of 20 ° C. for electrophoresis in a P / ACE MDQ molecular characterization system (Beckman Coulter). Eleven molecular size markers from 72 to 1353 bp were used. The nucleic acid detection limit (DL) for this method was 0.7 pg / ml.

The distribution and size of DNA fragments were measured based on size markers and relative densities of DNA bands, and were assigned to four different size categories ranging from <200 bp to> 1000 bp. FIG. 6 shows the capillary electrophoresis of the step (C) sample. Analysis shows that all detectable residual DNA in this sample is substantially less than 200 bp in length.

The average results from the five cultures are shown in the table below:

したがって、ＢＰＬ処理は、ＤＮＡ量の約１０倍減少を引き起こすだけでなく、長い配列から＜２００ｂｐの小さいフラグメントへ分布をシフトする。クロマトグラフィーおよび限外濾過工程を含む、工程（Ｂ）と（Ｃ）との間の、さらなる処理は、トータルＤＮＡレベルをさらに約７０倍低下させ、そして≧２００ｂｐの全ての検出可能なＤＮＡを除去した。

Therefore, BPL treatment not only causes a 10-fold reduction in the amount of DNA, but also shifts the distribution from long sequences to smaller fragments <200 bp. Further treatment between steps (B) and (C), including chromatography and ultrafiltration, further reduced total DNA levels by about 70-fold and removed all detectable DNA at ≧ 200 bp. bottom.

DNA Amplification Neoplasmic cell transformation is a phenomenon often associated with modified proto-oncogenes and / or modified tumor suppressor genes. Sequences from some such canine genes were analyzed by PCR before and after BPL treatment, ie at time points (A) and (B). In addition, DNA from uninfected MDCK cells was treated and tested. The proto-oncogenes tested were: H-ras and c-myc. The tumor suppressor genes tested were: p53; p21 / firewall; and PTEN. In addition, repeated SINE sequences were analyzed by PCR. A high copy count for SINE facilitates sensitive detection.

All samples were spiked with external control DNA (pUC19 fragment) to monitor sample preparation and PCR quality. In all experiments, constant amplification of the spike control was observed, which means that the residual hydrolyzed BPL and by-products present in the PCR mixture had no inhibitory effect on the assay, and sample preparation and sample preparation. It shows that it has ensured sufficient quality of PCR. Samples are serially diluted 10-fold prior to amplification until no PCR product can be detected, thereby exhibiting log reduction at the DNA level. The detection limit for the PCR assay is 55 pg.

PCR products were analyzed by agarose gel electrophoresis. FIG. 1 shows the results obtained in uninfected MDCK cells, and FIG. 2 shows the results obtained during virus culture. All six analyzed genes showed a strong amplification signal before BPL treatment, but signal intensity decreased after exposure to BPL. In all tested production lots, residual MDCK genomic DNA was reduced by at least 2 log values after BPL exposure.

FIG. 2 shows the results in step (B) prior to further purification, so the results are over-represented with the DNA present in the final bulk vaccine. However, due to the sensitivity when using the SINE sequence, PCR was also possible in step (C), in the final monovalent bulk. The results of this analysis are shown in FIG. DNA amplification in (A) and (B) is relatively similar for the SINE region, indicating that BPL is less active for smaller DNA sequences. However, even for these small sequences, there was a significant reduction in amplification signals for all of the samples analyzed. Comparison of PCR signal intensities at sample collection time points (B) and (C) reflects the reduction in residual DNA due to the intermediate purification step.

Total DNA levels of <1 ng per dose, and often <100 pg per dose, are expected in the final vaccine, inferred from gene-based analysis and directly based on SINE-based analysis.

Temperature BPL treatment during virus inactivation had two independent steps: (1) adding BPL to the virion-containing mixture at 2-8 ° C; then (2) raising the temperature to 37 ° C and BPL. Hydrolyzes. The effects of the two BPL steps on MDCK DNA inactivation and fragmentation were studied.

Purified genomic DNA from uninfected MDCK cells was treated at a final concentration of 0.05% (v / v) BPL for 16 hours at 2-8 ° C or 6.5 hours at 37 ° C or 50 ° C. .. DNA fragmentation will be checked later and the results are shown in FIG. These experiments reveal the activity of BPL on cellular DNA, but uninfected cells do not reflect the state of MDCK after viral growth, because cellular DNA is already highly due to apoptosis. This is because it is fragmented.

Differences between untreated and treated DNA on agarose gels at 2-8 ° C. are negligible, which means that DNA fragmentation by BPL can substantially occur during these conditions. Indicates that there is no such thing. In contrast, DNA is highly modified at both 37 ° C and 50 ° C, with higher temperatures leading to accelerated kinetics. DNA from untreated cells showed a relatively clear band on the agarose gel, accompanied by a light stain on the degradation products (FIG. 5, lane 3). However, after a brief BPL incubation at 37 ° C., MDCK DNA was smeared down from the gel slot and the band signal intensity was reduced (FIG. 5, lane 2). Longer incubation periods resulted in the disappearance of large genomic DNA molecules and enhanced fragmentation of MDCK DNA.
In a further experiment, BPL was first incubated with cells at 4 ° C. for 16 hours. In the first population, the temperature was raised to 37 ° C. for 2 hours; in the second population, the BPL was removed by centrifugal filtration and then the temperature was raised. Much lower residual DNA levels (lower than> 2 logs) were found in the first population.
Therefore, the DNA fragmentation observed during BPL treatment appears to occur predominantly during the BPL hydrolysis step at 37 ° C, rather than during the virus inactivation step at 2-8 ° C.

The present invention is described only as an example, and it is understood that modifications can be made while remaining within the scope and spirit of the invention.

Claims (10)

A method for preparing a cell culture product, which is a recombinant HA protein expressed in a cell culture of a mammal, bird or insect.
(I) Degrading residual functional cell culture DNA with β-propiolactone (BPL);
(Ii) With the step of treating with cetyltrimethylammonium bromide (CTAB) to solubilize the recombinant HA protein;
(Iii) A method comprising the step of isolating the recombinant HA protein. The method of claim 1, wherein the β-propiolactone (BPL) in step (i) is less than 0.1% β-propiolactone (BPL). The cell cultures are MDCK cells, Vero cells, PER. The method according to claim 1 or 2, which is selected from the group consisting of C6 cells and insect cells. A vaccine containing an immunogenic HA protein derived from influenza virus, or a preparation containing a recombinant HA protein, wherein the vaccine or preparation is (a) a residual functional cell culture of less than 10 ng / 0.5 mL. A vaccine or formulation comprising DNA, the average length of the residual cell culture DNA being less than 200 base pairs, and (b) neither ovoalbumin nor ovomucoid. The vaccine or formulation according to claim 4 , wherein the size of any residual cell culture DNA is measured by capillary gel electrophoresis. The vaccine or preparation according to any one of claims 4 to 5 , further comprising an oil-in-water emulsion adjuvant. The vaccine or preparation according to claim 6 , wherein the oil-in-water emulsion adjuvant has oil droplets having a submicron diameter. The vaccine or preparation according to claim 6 or 7 , wherein the oil-in-water emulsion adjuvant comprises squalene and polysorbate 80. The vaccine or preparation according to any one of claims 6 to 8 , wherein the oil-in-water emulsion adjuvant comprises squalene, tocopherol and polysorbate 80. The vaccine according to any one of claims 4 to 9 , wherein the vaccine comprises a divided virion or a purified surface antigen.

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